Protein tyrosine phosphatase inhibitor, preparation method and uses thereof

ABSTRACT

Disclosed in the present invention is a protein tyrosine phosphatase inhibitor. The preparation method therefor is: extracting the crude product from the Isaria Fumosorosea Wize solid or liquid fermentation broth using ethyl acetate, ethanol, methanol, or a mixed solvent of chloroform and methanol; separating the obtained extract using column chromatography on silica gel; and obtaining the target product. The inhibitor can be used to prepare pharmaceutical compositions for treating and preventing diabetes, obesity and cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/403,113 filed on Nov. 21, 2014, which is a U.S. NationalStage Application of International Application No. PCT/CN2013/075235filed on May 7, 2013, and published in Chinese as WO 2013/174207 A1 onNov. 28, 2013. This application claims priority to Chinese ApplicationNo. 201210158554.5 filed on May 22, 2012. The entire disclosures of theabove applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a protein tyrosine phosphataseinhibitor, to method for preparing the same, and to use thereof in themanufacture of a pharmaceutical composition for the treatment andprevention of diabetes, obesity and cancers.

BACKGROUND OF THE INVENTION

Protein tyrosine phosphatases (PTPs) belong to signal-transductionenzymes. They regulate cell growth, differentiation and metabolism bymodulating the phosphorylation level of intracellular tyrosine. PTPsalso regulate cell migration, genetic transcription, opening and closingof an ion channel, immune response, apoptosis and osteoblasticdevelopment. PTPs disorder may result in a variety of diseases such ascancer, diabetes, obesity and osteoporosis. To date, there has beenfound more than 130 gene-encoding PTPs in human genome. These highlyspecific PTPs are not only associated with the signal pathway of theinhibition and occurrence of cancers, but can also directly lead to theonset of different diseases in human due to their dysfunctions. Theaction mechanisms of PTPs associated with the onset of diseases as wellas the screening of their inhibitors are always the hot focus of theword.

PTPases which include a large family of transmembrane (receptor type)and intracellular (non-receptor type) enzymes are involved in regulationof a series of vital life processes. Several PTPases may affect theaction of insulin at the receptor or post-receptor stage in the insulinpathway. Currently, studies mainly focus on the research of SHP-2 andPTP1B.

PTP1B is the first protein tyrosine phosphatase that has beensuccessfully isolated. This enzyme is an intracellular enzyme of 37 kD.It anchors onto the endoplasmic reticulum via the 35 amino acidresidues-containing C-terminal. Cysteine at the 215^(th) position is thecatalytic active site of PTB1. Studies have shown that PTP1B blocks thesignal transduction pathway of insulin via Insulin Receptor (IR) andInsulin Receptor Substrate (IRS) and ultimately affects thetransportation of glucose from blood into cells, which represents as anelevated blood glucose. In addition, since PTP1B gene knockout mice areimmune to obesity, it can be determined definitely that PTP1B plays akey role in the pathogenesis of obesity. Consequently, PTP1B is a targetof the development of new medicaments for the treatment of diabetes andobesity.

Protein tyrosine phosphatase SHP2 [Src homology 2 (SH2) domaincontaining phosphotyrosinephosphatase 2] is a non-receptor type proteintyrosine phosphatase (PTP) which is encoded by PTPN11 gene and widelyexpressed in cytoplasm. SHP2 plays a key role in processes such as cellproliferation and differentiation by participating in a variety ofintracellular signal transductions, such as MAP kinase pathway, Jak-Statpathway, PI3 kinase pathway and other pathways. PTPN11 is identified asa proto-oncogene and also involves in the signal pathways of many otherproto-oncogenes as an important member. Hence, SHP2 serves as apotential drug target for the treatment of diseases such as Noonansyndrome and juvenile myelomonocytic leukemia, as well as other possiblerelevant tumor diseases.

Up to date, there is no report yet available in the prior art concerningthe extraction of a protein tyrosine phosphatase inhibitor from thefermentation product of Isaria Fumosorosea Wize.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is toprovide a novel protein tyrosine phosphatase inhibitor, method forpreparing the same and use thereof in the manufacture of apharmaceutical composition for the treatment and prevention of diabetes,obesity and cancers.

To solve the technical problem mentioned above, the technical solutionemployed in the invention is as follows:

A protein tyrosine phosphatase inhibitor, which is the following (a) or(b):

(a) a compound having the following structural formula:

(b) an isomer of the above compound or a pharmaceutically acceptablederivative thereof.

As a preferred technical solution of the invention, the pharmaceuticallyacceptable derivative thereof is a pharmaceutically acceptable salt,which maintains the biological effectiveness of said (a) and is an acidaddition salt or base addition salt commonly used in pharmacy andderived from non-toxic organic acids (e.g., carboxylic acid having from1 to 20 carbon atoms, sulfonic acid, amino acid, sulfamic acid, orsubstituents thereof), inorganic acids, organic bases or inorganicbases.

As a preferred technical solution of the invention, the inorganic acidsused for preparing said acid addition salt are hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid,phosphoric acid or nitric acid. The organic acids used for preparingsaid acid addition salt are p-toluenesulfonic acid, salicylic acid,methanesulfonic acid, oxalic acid, malic acid, succinic acid, lacticacid, fumaric acid. The basic agents used for preparing said baseaddition salt are hydroxides of alkali metal, alkali earth metal andquaternary ammonium; bicarbonates of alkali metal, alkali earth metaland quaternary ammonium; carbonates of alkali metal, alkali earth metaland ammonium; organic ammonium having from 1 to 30 carbon atoms;substituted pyridine and quinoline; particularly hydroxides of ammonium,potassium, sodium or quaternary ammonium, such as tetramethylammoniumhydroxide.

As a preferred technical solution of the invention, the pharmaceuticallyacceptable derivative thereof is in the form of ester or etherderivative; or it is incorporated with a functional group of aldehyde,acid or alcohol by oxidation of double bonds; or it is incorporated withsulfonic acid group in the ring; or it is incorporated with a chainhaving from 4 to 10 carbon atoms by Witting reaction; or part of or allof the double bonds on the side chain thereof are saturated; or it isincorporated on the side chain with one group or a combination of moregroups selected from the group consisting of alcohol, aldehyde, ether,thioether, sulfoxide, sulphone, halogen, carboxylic acid, sulfonic acid,amine, amide, sulfamide, saturated or unsaturated alkyl, cycloalkyl,fused alkyl and fused aryl group; or it is incorporated in one or tworings with one group or a combination of more groups selected from thegroup consisting of alcohol, aldehyde, ether, thioether, sulfoxide,sulphone, halogen, carboxylic acid, ester, sulfonic acid, amine, amide,sulfamide, saturated or unsaturated alkyl, cycloalkyl, fused alkyl andfused aryl.

As a preferred technical solution of the invention, the chainincorporated by Witting reaction carries OR¹, NR²R³ or halogen, whereinR¹, R² or R³ is H or alkyl having from 1 to 5 carbon atoms, and halogenis F, Cl, Br or I.

A method for preparing the above-mentioned protein tyrosine phosphataseinhibitor comprises: extracting a solid or liquid fermentation broth ofentomogenous fungus Isaria Fumosorosea Wize with ethyl acetate, ethanol,methanol, or a mixed solvent of chloroform and methanol; and separatingthe resulting extract by column chromatography on silica gel to obtainFumosorinone, namely, the protein tyrosine phosphatase inhibitor (a)according to the first aspect of the disclosure.

As a preferred technical solution for the above-mentioned preparationmethod, it comprises:

A. Fermentation and Cultivation: inoculating the Isaria Fumosorosea Wizestrain to a modified potato dextrose culture medium with an inoculationneedle; culturing the mixture in a shaker at 150 rpm and at 26° C. for 7days to obtain a seed broth; inoculating the seed broth to a riceculture medium and culturing under light at 26° C. for 30 days to obtaina fermented product; wherein, the said modified potato dextrose culturemedium is formulated by the following steps: dissolving 200 parts byweight of peeled potato, 20 parts by weight of dextrose, 3 parts byweight of KH₂PO₄, 1.5 parts by weight of MgSO₄, 0.1 parts by weight ofcitric acid, and 0.01 parts by weight of vitamin B₁ in 1000 parts byweight of sterile water, and adjusting pH to 6.5 and sterilizing at 121°C. for 20 minutes; and the rice culture medium is formulated by thefollowing steps: dissolving 80 parts by weight of rice in 100 parts byweight of sterile water, soaking for 12 hours, and then at natural pHsterilizing at 121° C. for 30 minutes;B. Extraction and Separation: extracting the fermented product bysoaking it in equal volume of ethyl acetate at room temperature for 48hours; evaporating to dryness by vacuum distillation with a rotatoryevaporator at 45° C. to obtain an extractum sample; thoroughly mixingsaid extractum sample with 1.2 times by weight of silica gel having amesh size of 100 to 200 under stirring, drying and grinding uniformly toobtain powders, rendering said extractum sample sufficiently absorbedonto the silica gel particles; then performing atmospheric columnchromatography on silica gel having a mesh size ranging from 300 to 400,and eluting gradiently with petroleum ether/ethyl acetate system inratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v); collectingthe eluents, evaporating by vacuum distillation at 45° C., concentratingand washing with an organic solvent; further subjecting the fractionobtained from the above elution section with 80:20 of petroleumether/ethyl acetate to the above-mentioned atmospheric columnchromatography on silica gel, and eluting gradiently again withpetroleum ether/ethyl acetate system in ratios of 100:0, 50:1, 20:1,15:1, 10:1 and 5:1 (v/v); collecting the eluents, evaporating by vacuumdistillation at 45° C. to obtain Fumosorinone at the 5:1 elutionsection, namely, to obtain the protein tyrosine phosphatase inhibitor(a) according to the first aspect of the disclosure.

As a preferred technical solution for the above-mentioned preparationmethod, it comprises:

A. Fermentation and Cultivation: inoculating the Isaria Fumosorosea Wizestrain to a modified potato dextrose culture medium with an inoculationneedle; culturing the mixture in a shaker at 150 rpm and at 26° C. for 7days to obtain a seed broth; inoculating the seed broth to a riceculture medium and culturing under light at 26° C. for 30 days to obtaina fermented product; wherein, the said modified potato dextrose culturemedium is formulated by the following steps: dissolving 200 parts byweight of peeled potato, 20 parts by weight of dextrose, 3 parts byweight of KH₂PO₄, 1.5 parts by weight of MgSO₄, 0.1 parts by weight ofcitric acid, and 0.01 parts by weight of vitamin B₁ in 1000 parts byweight of sterile water, and adjusting pH to 6.5 and sterilizing at 121°C. for 20 minutes; and the said rice culture medium is formulated by thefollowing steps: dissolving 80 parts by weight of rice in 100 parts byweight of sterile water, soaking for 12 hours, and then at natural pHsterilizing at 121° C. for 30 minutes;B. Extraction and Separation: extracting the fermented product bysoaking it in equal volume of ethyl acetate at room temperature for 48hours; evaporating to dryness by vacuum distillation with a rotatoryevaporator at 45° C. to obtain an extractum sample; thoroughly mixingsaid extractum sample with 1.2 times by weight of silica gel having amesh size of 100 to 200 under stirring, drying and grinding uniformly toobtain powders, rendering said extractum sample sufficiently absorbedonto the silica gel particles; then conducting atmospheric columnchromatography on silica gel having a mesh size ranging from 300 to 400,and eluting gradiently with petroleum ether/ethyl acetate system inratios of 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v); collectingthe eluents, evaporating by vacuum distillation at 45° C., concentratingand washing with an organic solvent; combining the eluents from eachelution section, performing the above-mentioned atmospheric columnchromatography on silica gel again, and eluting gradiently withpetroleum ether/ethyl acetate system in ratios of 100:0, 50:1, 20:1,15:1, 10:1 and 5:1 (v/v) again; collecting the eluents, evaporating byvacuum distillation at 45° C.; combining the fractions from each elutionsection again, and repeating the gradient elution with petroleumether/ethyl acetate in ratios of 100:0, 50:1, 20:1, 15:1, 10:1 and 5:1(v/v) to obtain Fumosorinone at the 5:1 elution section, namely, toobtain the protein tyrosine phosphatase inhibitor (a) according to thefirst aspect of the disclosure.

As a preferred technical solution for the above-mentioned preparationmethod, it comprises:

A. Fermentation and Cultivation: inoculating the Isaria Fumosorosea Wizestrain to a modified potato dextrose culture medium with an inoculationneedle; culturing the mixture in a shaker at 150 rpm and at 26° C. for 7days to obtain a seed broth; inoculating the seed broth to a riceculture medium and culturing under light at 26° C. for 30 days to obtaina fermented product; wherein, the said modified potato dextrose culturemedium is formulated by the following steps: dissolving 200 parts byweight of peeled potato, 20 parts by weight of dextrose, 3 parts byweight of KH₂PO₄. 1.5 parts by weight of MgSO₄, 0.1 parts by weight ofcitric acid, and 0.01 parts by weight of vitamin B₁ in 1000 parts byweight of sterile water, and adjusting pH to 6.5 and sterilizing at 121°C. for 20 minutes; and the said rice culture medium is formulated by thefollowing steps: dissolving 80 parts by weight of rice in 100 parts byweight of sterile water, soaking for 12 hours, and then at natural pHsterilizing at 121° C. for 30 minutes;B. Extraction and Separation: extracting the fermented product bysoaking it in equal volume of ethyl acetate at room temperature for 48hours; evaporating to dryness by vacuum distillation with a rotatoryevaporator at 45° C. to obtain an extractum sample; thoroughly mixingsaid extractum sample with 1.2 times by weight of silica gel having amesh size of 100 to 200 under stirring, drying and grinding uniformly toobtain powders, rendering said extractum sample sufficiently absorbedonto the silica gel particles; then performing atmospheric columnchromatography on silica gel having a mesh size ranging from 300 to 400,eluting gradiently with petroleum ether/ethyl acetate system in ratiosof 100:0, 95:5, 90:10, 80:20, 60:40 and 50:50 (v/v); collecting theeluents, evaporating by vacuum distillation at 45° C., concentrating andwashing with an organic solvent; further subjecting the fractionobtained from the above elution section with 80:20 of petroleumether/ethyl acetate to the above-mentioned atmospheric columnchromatography on silica gel, and eluting gradiently withchloroform/methanol in ratios of 100:0, 95:5 and 90:10 (v/v) to obtainFumosorinone at the 90:10 elution section, namely, to obtain the proteintyrosine phosphatase inhibitor (a) according to the first aspect of thedisclosure.

Use of the above-mentioned protein tyrosine phosphatase inhibitor in themanufacture of a pharmaceutical composition for the treatment andprevention of diabetes, obesity and cancers.

The beneficial effects produced by the above-mentioned technicalsolutions lie in that: the inventors have verified the actual effects ofcompound (a) of the invention by experiments, in particular as follows:

{circle around (1)} In-vitro Inhibition Experiment of compound (a) ofthe invention on protein tyrosine phosphatase PTP1B.

Enzyme: human PTP1B was cloned from cDNA library by conventionalmolecular biological methods. The cDNA sequence was identical with thatof the public human PTP1B (Accession No. M33689). The protein wasexpressed and purified from E. coli, as described in Barford D. et al.,J. Mol Biol (1994) 239, 726-730. A phosphorylated tyrosine peptide basedon the amino acid sequence at tyrosine autophosphorylation site 1146(TRDI(Py)E) of the insulin receptor was used as the substrate. Reactionconditions were as follows: PTP1B (05-2 Nm) was incubated with thecompound for 15 minutes in a buffer containing 37.5 nM Bis-Tris (at pH6.2), 140 nM NaCl, 0.05% BSA and 2 Mm DTT. The reaction was initiatedwith the addition of 50 μM substrate under room temperature (22-25□) andterminated with KOH. Malachite green was used to detect the amount ofphosphate. The experimental result shows that: the inhibitory activity(IC₅₀) of Fumosorinone, namely the protein tyrosine phosphataseinhibitor of the invention, on protein tyrosine phosphatase PTP1B is0.42 μg/ml.

{circle around (2)} In-vitro Inhibition Experiment of compound (a) ofthe invention on protein tyrosine phosphatase SHP2.

Enzyme: human SHP2 was cloned from cDNA library by conventionalmolecular biological methods. The cDNA sequence was identical with thatof the public human SHP2 (Accession No. M002834). The protein wasexpressed and purified from E. coli, as described in Ute Dechert. etal., J. Biol Chem, 1994, 269(8): 5602-5611. SHP2 hydrolyzed thephosphate group of pNPP (p-nitrophenyl phosphate), and the product pNP(p-nitrophenol) showed absorbance at 405 nm. The absorbance valueincreased in positive proportion to the time of enzymatic reaction.Hence, it was possible to reflect initial rate of enzymatic reactionaccording to the slope of the increased absorbance curve and determinethe activity of SHP2. The enzymatic reaction system components was asfollows: a buffer (50 mM Bis-Tris at pH 6.0, 2 mM DTT, 2 mM EDTA,GST2-SHP2, and 2 mM pNPP). The enzymatic activity was calculated bydynamically measuring the absorbance value at the wavelength of 405 nmunder room temperature after uniformly mixing of the reaction system.The experimental result shows that: the inhibitory activity (IC₅₀) ofFumosorinone, namely the protein tyrosine phosphatase inhibitor of theinvention, on protein tyrosine phosphatase SHP2 is 0.90 μg/ml.

{circle around (3)} In-vivo verification of the hypoglycemic effect ofcompound (a) of the invention.

An animal model of experimental diabetes was established by damagingpancreas or islet cells, thereby leading to insulin-deficiency, or byusing an antagonist against the action of insulin, resulting inexperimental diabetes or experimental hyperglycemia. Alloxan-induceddiabetic model is the most common model in the current research on humandiabetes, and alloxan-induced diabetic mouse model was used to test theportion of hypoglycemic activity among the activities of the targetcompound.

Establishment of the experimental animal model of diabetes: Male mice(of Kunming species) were purchased and fed for three days, and theywere adapted to the environment before experiment. Mice of similarweights (26±2 g) were fasted for 16 to 18 hours and thenintraperitoneally injected with 1% of alloxan saline solution (preparedbefore use) one time or multiple times in succession in a dose of 200mg/kg. Multiple injections were performed at an interval of 3 days andwere performed consecutively for 4 times with an alloxan solution in thesame dose. Blood samples were taken from tails of the mice undernon-fasting state at 48 hours after injection, and the blood glucosevalue was measured. The standard value was set as blood glucosevalue >12.0 mmol/l. Mice whose blood glucose values failed to return tonormal value after one week were identified as mouse model of diabetesand defined as the diabetic group. Mice in the normal control group wereintraperitoneally injected with saline in the same dose.

Grouping and treatment of the experimental animals: there were sixgroups of mouse model of diabetes, namely, 10 mice of the normal controlgroup, 10 mice (with blood glucose value >12 mmol/1) of the modelcontrol group, 10 mice of the positive control group and 30 mice of theexperimental groups (i.e. groups of target compound in low, medium andhigh doses). After being classified into each group, mice wereadministered by gavage. Mice from the normal control group and the modelcontrol group were administered with normal saline. Mice from thepositive control group were daily administered by gavage with 50 mg/kgof gliclazide. Mice from said three experimental groups wererespectively administered by gavage with a saline solution of the targetcompound in different doses (12.5 mg/kg, 25 mg/kg, and 50 mg/kg), twicea day (one before noon and the other after noon), each time in halfdose.

Measurement of blood glucose: blood samples were taken from the tailvein of mice that had been fasted for 16 to 18 hours, respectively atthe 5^(th) day, 10^(th) day and 15^(th) day after administration, andthe blood glucose was measured.

Experimental result: see table 1. As compared with those from the modelcontrol group, 5 days after administration by gavage, mice from therespective drug-administered groups showed decreased blood glucosevalues.

Table 1. Effect of the target compound on the blood glucose of mice withalloxan-induced diabetes.

TABLE 1 blood glucose blood glucose blood glucose blood glucoseconcentration concentration concentration concentration dose (mmol/L)(mmol/L) (mmol/L) (mmol/L) Group (mg/kg) 0 d 5 d 10 d 15 d Normalcontrol — 4.23 ± 4.6 ± 5.18 ± 4.28 ± group 0.48aA 0.45aA 1.83aB 0.89aAModel control — 23.8 ± 24.6 ± 25.1 ± 25.64 ± group 2.36bA 1.35bA 3.34bA1.56bA Positive control 50 24.4 ± 3.21 21.2 ± 19.3 ± 15.4 ± group bA2.89cB 1.12cC 2.26cD Low-dose 12.5 23.8 ± 22.8 ± 18.4 ± 15.3 ± group4.32bA 2.68cA 1.38cB 2.21cC Medium-dose 25 24.1 ± 20.6 ± 17.8 ± 12.9 ±group 3.35bA 1.87cB 2.38cC 2.72dD High-dose 50 23.6 ± 12.16 ± 10.34 ±6.32 ± 3.2eC group 2.84bA 4.36cA 2.31dBNote: different lowercase letters in the same column indicated that theblood glucose values of mice from different groups were significantlydifferent at the level of P<0.05; different uppercase letters in thesame line indicated that the blood glucose values of mice at differenttimes after administration were significantly different at the level ofP<0.05.{circle around (4)} According to the common knowledge of pharmacy,compound (a) of the invention can produce a variety of derivativeshaving similar effects by simple chemical reactions. The actual effectsof these derivatives are expectable to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by the following examples. All startingmaterials and equipment employed in the invention are conventional andcommercially available and can be directly purchased from the market.

Example 1 Preparation of the Product

The Isaria Fumosorosea Wize (=Paecilomyces fumosoroseus (Wize)A.H.S.Br.&G.Sm) used in the invention was collected from Baoding city,Hebei Province; and the strain was kept at the College of Life Science.Hebei University. For specific source, please refer to the DisclosureForm of the Origin of Genetic Resources.

A. Fermentation and Cultivation: Isaria Fumosorosea Wize strain wasinoculated with an inoculation needle into a 500 ml Erlenmeyer flaskcontaining 100 ml of modified potato dextrose culture medium (formulatedby dissolving 200.0 g of peeled potato, 20.0 g of dextrose, 3.0 g ofKH₂PO₄. 1.5 g of MgSO₄, 0.1 g of citric acid, and 10.0 mg of vitamin B₁in 1 l of sterile water, pH 6.5 and sterilizing at 121° C. for 20minutes). The mixture was cultured in a shaker at 150 rpm and at 26° C.for 7 days, to obtain a seed broth, 10 ml of the seed broth wasinoculated into a 500 ml Erlenmeyer flask containing rice culture medium(formulated by dissolving 80 g of rice in 100 ml of sterile water,soaking for 12 hours, and then at natural pH sterilizing at 121° C. for30 minutes). There were 50 Erlenmeyer flasks in total. The mixture wasfurther cultured under light at 26° C. for 30 days.B. Extraction and Separation: the fermented product was extracted bysoaking with equal volume of ethyl acetate at room temperature for 48hours, and evaporated to dryness by vacuum distillation with a rotatoryevaporator at 45° C. for three times, to obtain 200 g of extractumsample. Said extractum sample was thoroughly mixed with 240 g of silicagel (with a mesh size of 100 to 200) under stirring, dried, grounduniformly to obtain powders, rendering said extractum samplesufficiently absorbed onto the silica gel particles. The mixture wasthen subjected to atmospheric column chromatography on silica gel (witha mesh size ranging from 300 to 400), and eluted gradiently withpetroleum ether/ethyl acetate system (100:0, 95:5, 90:10, 80:20, 60:40and 50:50 (v/v)). The eluents were collected in 500 ml triangularflasks, subjected to vacuum distillation at 45° C., concentrated andwashed with a small amount of organic solvent. The resulting sampleswere placed in 5 ml flaskets and the flaskets were numbered and labeled,37 g of the fraction obtained from the elution section with 80:20 ofpetroleum ether/ethyl acetate was further subjected to atmosphericcolumn chromatography on silica gel under gradient elution withpetroleum ether/ethyl acetate system (100:0, 50:1, 20:1, 15:1, 10:1 and5:1 (v/v)). The eluents were collected in 200 ml triangular flasks andsubjected to vacuum distillation at 45° C., to obtain 10 g ofFumosorinone at the 5:1 elution section, namely, to obtain the proteintyrosine phosphatase inhibitor (a).

Example 2 Preparation of the Product

This example differed from example 1 in the following procedures: theextractum sample, after being sufficiently adsorbed on the silica gelparticles, was subjected to atmospheric column chromatography on silicagel (with a mesh size ranging from 300 to 400), and eluted gradientlywith petroleum ether/ethyl acetate system in ratios of 100:0, 95:5,90:10, 80:20, 60:40 and 50:50 (v/v). The eluents were collected,subjected to vacuum distillation at 45° C., concentrated and washed withan organic solvent. The eluents from each elution section were combinedand subjected to the above-mentioned atmospheric column chromatographyon silica gel again, and eluted gradiently again with petroleumether/ethyl acetate system in ratios of 100:0, 50:1, 20:1, 15:1, 10:1and 5:1, (v/v). The eluents were collected and subjected to vacuumdistillation at 45° C. The eluents from each elution section werecombined again and further subjected to the gradient elution processwith petroleum ether/ethyl acetate in ratios of 100:0, 50:1, 20:1, 15:1,10:1 and 5:1 (v/v), to obtain Fumosorinone at the 5:1 elution section,namely, to obtain the protein tyrosine phosphatase inhibitor (a).

The yield of product was advantageously further increased by eluting theproduct in such manner.

Example 3 Preparation of the Product

This example differed from example 1 in the following procedures: theextractum sample, after being sufficiently adsorbed on the silica gelparticles, was subjected to atmospheric column chromatography on silicagel (with a mesh size ranging from 300 to 400), and eluted gradientlywith petroleum ether/ethyl acetate system in ratios of 100:0, 95:5,90:10, 80:20, 60:40 and 50:50, (v/v). The eluents were collected,subjected to vacuum distillation at 45° C., concentrated and washed withan organic solvent. The fraction obtained from the above elution sectionwith 80:20 of petroleum ether/ethyl acetate was further subjected to theabove-mentioned atmospheric column chromatography on silica gel, andeluted gradiently with chloroform/methanol system in ratios of 100:0,95:5 and 90:10 (v/v), to obtain Fumosorinone at the 90:10 elutionsection, namely, to obtain the protein tyrosine phosphatase inhibitor(a).

Example 4 Determination of the Structure of the Product

The structure of Fumosorinone was determined according to the spectraldata of mass spectrum. NMR spectrum and the like.

Data of UV spectrum: UV (MeOH) λ_(max) nm: 367 (6.87), 265 (7.20), 209(7.19).

Data of Infrared spectrum: IRν^(KBr) _(max)cm⁻¹: 3267, 2960, 1643, 1516,1446, 1268, 1218, 837, 758.

Data of Mass Spectrum: HR-ESI-MS m/z: 500.24100 ([M+Na]⁺, 500.24074calcd. for C₂₉H₃₅NNaO₅).

For NMR data, see Table 2.

TABLE 2 ¹H-(600 MHz) and ¹³C-NMR (150 MHz) Data of Fumosorinone inCD₃OD¹ δ (H) δ (C) HMBC(H → C) C(2) 158.6 C(3) 111.1 C(4) 166.5 C(5)114.9 H—C(6) 7.87 (s) 137.9 1′, 2, 4, 5 C(7) 199.8 C(8) 137.1 H—C(9)6.94 (d, J = 9.8) 141.8 7, 10, 24 H—C(10) 6.67(m) 128.2 11 H—C(11)6.67(m) 142.9 10 H—C(12) 6.41 (dd, J = 15.2, 127.6 13 9.3) H—C(13) 6.45(d, J = 15.3) 143.4 12, 15 C(14) 134.1 H—C(15) 5.39 (d, J = 9.8) 144.213, 16, 17 H—C(16) 2.68 (m) 31.9 15 CH₂(17) 1.16 (m), 1.34 (m) 46.1 18H—C(18) 1.34 (m) 33.7 17, 19, 21 CH₂(19) 1.16 (m), 1.34 (m) 31.3 18, 20Me(20) 0.88 (m) 11.7 18, 19 Me(21) 0.88 (m) 19.6 17, 18, 19 Me(22) 0.98(d, J = 6.6) 21.8 15, 16, 17 Me(23) 1.84 (s) 12.7 13, 14, 15 Me(24) 2.06(s) 12.4 7, 8, 9 C(1′) 125.0 H—C(2′) 7.32 (d, J = 8.6) 131.7 5, 2′, 4′H—C(3′) 6.84 (d, J = 8.6) 116.4 1′, 4′ C(4′) 159.1 H—C(5′) 6.84 (d, J =8.6) 116.4 1′, 4′ H—C(6′) 7.32 (d, J = 8.6) 131.7 5, 4′, 5′

It can be determined from the above spectral data that Fumosorinone,i.e., the protein tyrosine phosphatase inhibitor (a) of the invention,has the following structure:

Example 5 Use of the Product

According to the experiments described above, the use of the proteintyrosine phosphatase inhibitor (a) of the invention lies in themanufacture of a medicament, in particular a pharmaceutical compositionfor the treatment and prevention of diabetes, obesity and cancers. Whenit is used as a medicament, this compound can either be used directly orin the form of a pharmaceutical composition. The pharmaceuticalcomposition may contain 0.1% to 99%, preferably 0.5% to 90% of thecompound of the invention, and the balance of pharmaceuticallyacceptable carriers or excipients which are non-toxic and inert to humanand animals. The pharmaceutically acceptable carriers or excipients maybe one or more selected from the group consisting of fillers of solid,semi-solid and liquid diluents, or auxiliaries for drug products. Thepharmaceutical composition is administered in the form of dosage perunit body weight. The medicament of the invention may be administeredorally or by injection (intravenous or intramuscular injection). Oraladministration may employ solid or liquid formulations thereof, such aspowders, tablets, dragees, capsules, solutions, syrups and droppingpills. Injection administration may employ solid or liquid formulationsthereof, such as powder injections and solution injections.

In the case of parenteral administration for human body, in order toachieve optimal effects, it is advantageous to employ an amount rangingfrom 0.1 to 10,000 μg/kg, preferably from 1 to 1,000 μg/kg, and inparticular from 1 to 80 μg/kg body weight. In the case of oraladministration, the amount is ranging from 0.2 to 10 mg/kg, preferably0.6 to 6 mg/kg, and in particular from 1 to 5 mg/kg body weight.Nevertheless, depending on the body weight, administration route,individual response to the active compound, formulation type, and thetime and intervals of administration, an amount deviated from saidranges may be necessary.

Example 6 Preparation of the Medicament

The compound Fumosorinone prepared according to example 1, was mixedwith excipients at a weight ratio of 1:1 of the compound in crystal formto the excipients, and then granulated and compressed into tablets; or

The compound Fumosorinone prepared according to example 1, wasformulated into capsules by conventional methods for preparing capsules;or

The compound Fumosorinone prepared according to example 1, was mixedwith excipients at a weight ratio of 1:2 of the compound in crystal formto the excipients, and then granulated and compressed into tablets; or

The compound Fumosorinone prepared according to example 1, was mixedwith excipients at a weight ratio of 1:3 of the compound in crystal formto the excipients, and then granulated and compressed into tablets.

Example 7 Preparation of the Medicament

Tablet: Fumosorinone 100 mg Starch 100 mg 17% of Corn syrup appropriateamount Magnesium stearate appropriate amount

Example 8 Preparation of the Medicament

Capsule: Fumosorinone 100 mg Starch 100 mg Magnesium stearateappropriate amount

Preparation method: Fumosorinone was mixed with auxiliaries, and thensieved and thoroughly mixed in a proper container. The resulting mixturewas placed into hard gelatin capsules.

Example 9 Preparation of the Medicament

Ampoule: Fumosorinone 50 mg

Preparation method: Fumosorinone was dissolved in 2 ml of propyleneglycol. After filtration, the resulting solution was placed intoampoules under sterile condition.

In addition, the protein tyrosine phosphatase inhibitor (a) of theinvention may further be an isomer of the above-mentioned compound (a)or pharmaceutically acceptable derivative thereof. The pharmaceuticallyacceptable derivative is a commonly used pharmaceutical derivative whichmaintains the biological effectiveness of said compound (a). Theselectable range and preparation method for such pharmaceuticallyacceptable derivative are common knowledge in the field of pharmacy. Theillustrations as described above are only proposed as implementabletechnical solutions of the invention and would not serve as a singlelimitation to the technical solutions per se.

What is claimed is:
 1. A method for treating diabetes, obesity andcancers in a subject in need thereof, the method comprising:administering a composition comprising a protein tyrosine phosphataseinhibitor and a pharmaceutically acceptable carrier to the subject,wherein the composition is administered in an effective amount, whereinthe protein tyrosine phosphatase inhibitor is the following (a) or (b):(a) a compound having the following structural formula:

(b) an isomer of compound (a) or a pharmaceutically acceptablederivative thereof.
 2. The method according to claim 1, wherein thecomposition is parenterally administered in an amount ranging from 0.1to 10.000 μg/kg body weight of the subject.
 3. The method according toclaim 1, wherein the composition is parenterally administered in anamount ranging from 1 to 1.000 μg/kg body weight of the subject.
 4. Themethod according to claim 1, wherein the composition is parenterallyadministered in an amount ranging from 1 to 80 μg/kg body weight of thesubject.
 5. The method according to claim 1, wherein the composition isorally administered in an amount ranging from 0.2 to 10 mg/kg bodyweight of the subject.
 6. The method according to claim 1, wherein thecomposition is orally administered in an amount ranging from 0.6 to 6mg/kg body weight of the subject.
 7. The method according to claim 1,wherein the composition is orally administered in an amount ranging from1 to 5 mg/kg body weight of the subject.
 8. The method according toclaim 1, wherein the composition is in the form of powder, tablet,dragee, capsule, solution, syrup or dropping pill for oraladministration.
 9. The method according to claim 1, wherein thecomposition is in the form of a powder injection or a solution injectionfor injection administration.
 10. The method according to claim 1,wherein the pharmaceutically acceptable derivative thereof is apharmaceutically acceptable salt, which maintains the biologicaleffectiveness of said compound (a) and is an acid addition salt or baseaddition salt commonly used in pharmacy and derived from non-toxicorganic acids, inorganic acids, organic bases or inorganic bases. 11.The method according to claim 10, wherein the inorganic acids forpreparing said acid addition salt are hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid ornitric acid; the organic acids used for preparing said acid additionsalt are p-toluenesulfonic acid, salicylic acid, methanesulfonic acid,oxalic acid, malic acid, succinic acid, lactic acid, or fumaric acid;and the basic agents for preparing said base addition salt arehydroxides of ammonium, potassium, sodium or quaternary ammonium. 12.The method according to claim 1, wherein the pharmaceutically acceptablederivative of compound (a) is in the form of an ester or etherderivative; or it is incorporated with a functional group of aldehyde,acid or alcohol by oxidation of double bonds; or is incorporated with asulfonic acid group in the ring; or it is incorporated with a chainhaving from 4 to 10 carbon atoms by a Witting reaction.
 13. The methodaccording to claim 12, wherein the chain incorporated by the Wittingreaction carries OR¹, NR²R³ or halogen, wherein R¹, R² or R³ is H oralkyl having from 1 to 5 carbon atoms, and the halogen is F, Cl, Br orI.